The red blood cell (RBC) membrane skeleton is composed principally of short F-actin filaments cross-linked by a2B2 spectrin tetramers with the assistance of protein 4.1 R. Actin and 4.1 R bind to the actin binding domain (BSpABD) at the tail of the spectrin B chain. The adjacent end of a-spectrin, called the EF hand domain (aSpEF), has been considered inert;however, the sph(1J) mouse, which has very unstable RBC membranes, makes a mutant a-spectrin that lacks the last 13 amino acids (aSpEFAC13), showing that the domain has an important function in spectrin-actin binding. 1) We will determine what binds to aSpEF, using pull-down assays or photoactivatable cross-linking, and extracts containing all RBC membrane proteins. Initial studies show that aSpEF but not aSpEFAC13 binds to protein 4.2 in a Ca (2+)-dependent manner. We will characterize this and other aSpEF-protein interactions and investigate whether post-translational modifications or other factors affect the binding. 2) We will ascertain whether binding site fragments that interfere with aSpEF interactions decrease membrane stability measured in the ektacytometer. 3) We will prepare a series of mini-spectrins consisting of the BSpABD domain and variants of the aSpEF domain attached by the last 4 spectrin repeats in each chain. The binding of these mini-spectrins to F actin and the roles of 4.1 R, 4.2 and other aSpEF binding proteins will be assessed. 4) Finally, we will identify proteins adjacent to 4.1 R or 4.2 on the membrane, or adjacent to aSpEF in actin junctional complexes using photoactivatable crosslinkers, and we will visualize the structure of the normal unstretched membrane skeleton and the skeletal defects in 4.2(-) and sph (1J) mice using high resolution EM and immunoEM. These studies will provide new information about the molecular junctions that form the core of the membrane skeleton and will illuminate the functions of the previously little studied EF hand domain. Since plasma membranes of all cells and membranes of many organelles have spectrin skeletons, these studies will be of broad interest in cell biology.